1Division of Biology, California Institute of Technology, Pasadena, California, United States of America.

Abstract

Genetically modified mice carrying engrafted human tissues provide useful models to study human cell biology in physiologically relevant contexts. However, there remain several obstacles limiting the compatibility of human cells within their mouse hosts. Among these is inadequate cross-reactvitiy between certain mouse cytokines and human cellular receptors, depriving the graft of important survival and growth signals. To circumvent this problem, we utilized a lentivirus-based delivery system to express physiologically relevant levels of human interleukin-7 (hIL-7) in Rag2-/-gammac-/- mice following a single intravenous injection. hIL-7 promoted homeostatic proliferation of both adoptively transferred and endogenously generated T-cells in Rag2-/-gammac-/- Human Immune System (HIS) mice. Interestingly, we found that hIL-7 increased T lymphocyte numbers in the spleens of HIV infected HIS mice without affecting viral load. Taken together, our study unveils a versatile approach to deliver human cytokines to HIS mice, to both improve engraftment and determine the impact of cytokines on human diseases.

HIS mouse T cells express the IL-7Rα and exhibit increased viability in response to hIL-7 in vitro.

a. Human immune cell populations were analyzed in lymphoid tissues from 20 week old HIS mice by flow cytometry to determine the extent of T and B cell engraftment. b. Peripheral blood from HIS mice or normal human donors was stained with either fluorophore-conjugated isotype control (clear histogram) or anti-hIL-7Ra antibody (grey histograms) and analyzed by flow cytometry. c. HIS mouse splenocytes (1×106) or lymph nodes cells (5×105) were cultured in increasing amounts of hIL-7 for 7 days. Cells were stained with antibodies against CD3, CD4 or CD8 as well as 7-AAD and analyzed by flow cytometry to quantify the number of live cells of each subtype.

Schematic of the lentiviral vector used to deliver hIL-7 or luciferase. b. Expression of luciferase was assayed using Xenogen imaging two months after intravenous injection of Rag2-/-γc-/- mice with 2×108 infectious units of luciferase expressing lentiviral vector. Localized expression from spleen (SP), bone marrow (BM), and liver (LV) are illustrated. c. Expression of hIL-7 was assayed for six months in the serum of Rag2-/-γc-/- mice following a single intravenous injection of either 1×108 or 4×108 infectious units of IL-7 expressing lentiviral vectors. Four mice were used per group, and the average and SEM are shown.

a. Serum concentrations of hIL-7 detected by ELISA three weeks after intravenous administration of 9×107 or 1.7×108 IU of lentivirus expressing either luciferase or hIL-7. b. The percentage of CD3+, CD4+ or CD8+ T cells of live splenocytes following one week post transfer of 2×107 CFSE labeled human PBMCs into Rag2-/-γc-/- mice from A. c. Average mean fluorescence intensity (MFI) of CFSE measured by flow cytometry in T-cell subsets quantified in B. Four mice were used per group, and the average and SEM are shown. d. Representative histograms showing CFSE loss by CD3+, CD4+ or CD8+ adoptively transferred T cells from mice receiving the control vector, low dose hIL-7 or high dose hIL-7.

Lentiviral vector delivery of hIL-7 to HIS mice improves T cell levels in the peripheral blood.

a. Total human CD45+ cell engraftment in peripheral blood of cohort of mice used in this experiment prior to separation into treatment groups. b. Serum concentrations of hIL-7 detected by ELISA at 18 weeks of age in mice receiving intravenous low (1×108 IU) or high (5×108 IU) dose lentivirus expressing either luciferase or hIL-7. c. HIS mice were injected with 1×108 (low dose) or 5×108 (high dose) hIL-7 or luciferase expressing lentiviral vectors at 8 weeks of age (indicated by black arrow). The percentages of CD3+ and CD19+ peripheral blood cells (as a percentage of total human CD45+ cells) were determined from 8 to 18 weeks of age by flow cytometry. 5–7 mice were used per group, and the average and SEM are shown.d. Proportion of CD3 and CD19 expressing cells in peripheral blood of a representative luciferase or hIL-7 expressing HIS mice at 18 weeks of age as compared to a normal human donor. e. For each mouse group, human cell engraftment was determined by calculating the percentage of human CD45+ cells of total CD45+ cells. The percentage of CD3+ CD4+ and CD3+CD8+ T cells in the peripheral blood was also determined. CD3+ naïve (CD27+CD45RA+), effector (CD27+CD45RA-) and memory (CD27-CD45RA-) phenotype T cells were quantified by flow cytometry.

Lentiviral vector delivery of hIL-7 improves T cell levels in the spleens and lymph nodes of HIS mice, and increases BCL2 expression.

Spleens were removed from hIL-7 (low dose group) or luciferase expressing mice and weighed. b. Splenic sections were H&E stained (scale bar = 1 mm). c. Spleens and lymph nodes were processed into single cell suspensions and flow cytometry was used to analyze the percentage of human CD3+ versus CD19+ cells in the different groups. CD3+ cells were analyzed to quantify CD4+ and CD8+ subsets. d. Absolute cell numbers for the indicated lineages were determined using splenocytes from hIL-7 expressing or control mice. e. Serial splenic sections were stained with antibodies against human CD3 (scale bar = 100 µm) or CD20 (scale bar = 100 µm), and another set with CD3 (scale bar = 100 µm) and BCL2 (scale bar = 100 µm). f. Serum from both low and high dose hIL-7 expressing mice or luciferase controls was assayed to determine the concentrations of total IgM or total IgG.

IL-7 or luciferase expressing HIS mice were infected with the HIV strain JR-CSF and peripheral blood was assayed by flow cytometry to determine the percentage of CD4+ of CD3+ human T cells. b. The HIV genome copy number in the peripheral blood plasma from the two groups of mice was quantified following 6 weeks of infection. c. The human CD45+, CD3+, CD4+, CD8+ and CD19+ splenocyte numbers were quantified by FACS 6 weeks after infection by HIV. d. Fixed Spleen sections from HIV infected luc and hIL-7 mice were stained for p24 (left, scale bar = 100 µm). The number of p24 positive cells in a given area (1 mm2) of a lymphoid follicle was determined (right). Data represent 8–9 mice per group, and the average and SEM are shown.